Comparison of Bacterial Diversity within the Coral Reef Sponge, Axinella corrugata, and the Encrusting Coral Erythropodium caribaeorum

We compared the Caribbean reef sponge, Axinella corrugata, with the Caribbean reef coral, Erythropodium caribaeorum for differences in their resident microbial communities. This cursory survey of bacterial diversity applied 16S rRNA gene sequences. Over 100 culture-independent sequences were generated from five different Axinella 16S rRNA libraries, and compared with 69 cultured isolates. The cultureindependent 16S rDNA clones displayed a higher diversity of Proteobacteria, including “uncultured” or “unknown” representatives from the Deltaproteobacteria. Arcobacterium, and Cyanobacteria were also found. We have also confirmed that Axinella sponges appeared to host specific microbial symbionts, similar to the previously identified clones termed “OSO” environmental samples. In contrast, seawater samples near Axinella were dominated by Pseudoalteromonas. Adjacent sediment samples yielded clones of Planctomycetacea, Proteobacteria, sulfate-reducing Desulfovibrio spp, and other Deltaproteobacteria. Anaerobe-like 16S rRNA sequences were detected after the oxygen supply to one Axinella sample was deliberately curtailed to assess temporal changes in the microbial community. E. caribaeorum yielded more Betaproteobacteria relative to Axinella 16S libraries, and also included the Gammaproteobacteria genus Spongiobacter. However, Axinella-derived microbes appeared phylogenetically deeper with greater sequence divergences than the coral. Overall this study indicated that marine microbial community diversity can be linked to specific source hosts and habitats

Comparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp.

Marine invertebrates such as soft corals are important sources of secondary metabolites with promising biomedical applications and commercial value. RNA isolation in conjunction with reverse-transcriptase polymerase chain reaction (RT-PCR) are valuable tools utilized to study the molecular elements involved in secondary metabolite production and functional genomics. Two total RNA extraction protocols were compared using fresh tissue and flash frozen preparations from the coral Pseudopterogorgia elisabethae and from its symbiont Symbiodinium sp. isolated using RNeasy minicolumns (Qiagen\uae) and Trizol reagent (Invitrogen\uae). In general, higher yields were obtained by using Trizol reagent when compared to RNeasy. No significant differences were observed in RNA yield when live or flash frozen tissue was used. However, flash frozen holobiont tissue isolated by Trizol resulted in the highest RNA yield of all preparations analyzed. To conclude, both protocols are suitable for RNA isolation. Trizol is recommended if higher yields are the primary concern, but RNeasy is recommended if time is an issue

Suppression Subtractive Hybridization PCR Isolation of cDNAs from a Caribbean Soft Coral

Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non- housekeeping genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various hypothetical protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 - FL512331, and GH611838]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17% of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution

Suppression subtractive hybridization PCR isolation of cDNAs from a Caribbean soft coral

Transcriptomic studies of marine organisms are still in their infancy. A partial, subtracted expressed sequence tag (EST) library of the Caribbean octocoral Erythropodium caribaeorum and the sea fan Gorgonia ventalina has been analyzed in order to find novel genes or differences in gene expression related to potential secondary metabolite production or symbioses. This approach entails enrichment for potential non-\u201chousekeeping\u201d genes using the suppression subtractive hybridization (SSH) polymerase chain reaction (PCR) method. More than 500 expressed sequence tags (ESTs) were generated after cloning SSH products, which yielded at least 53 orthologous groups of proteins (COGs) and Pfam clusters, including transcription factors (Drosophila Big Brother), catalases, reverse transcriptases, ferritins and various \u201chypothetical\u201d protein sequences. A total of 591 EST sequences were deposited into GenBank [dbEST: FL512138 - FL512331, GH611838, and HO061755-HO062154]. The results represent proof of concept for enrichment of unique transcripts over housekeeping genes, such as actin or ribosomal genes, which comprised approximately 17% of the total dataset. Due to the gene and sequence diversity of some ESTs, such sequences can find utility as molecular markers in current and future studies of this species and other soft coral biogeography, chemical ecology, phylogenetics, and evolution

Blockade of Mast Cell Activation Reduces Cutaneous Scar Formation

Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1a, IL-1b, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase b1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound

Stimulant Vs. Exercise Use for Symptomatic Relief in Postural Orthostatic Tachycardia Syndrome

Abstract: Introduction: Postural orthostatic tachycardia syndrome (POTS) is a form of orthostatic intolerance prominent in young females, and can be difficult to diagnose and treat. POTS is defined as a clinical syndrome of orthostatic intolerance that has a heart rate (HR) increase of 30 beats per minute (BPM) or more, with a standing HR of \u3e120 bpm in the context of a 10 minute standing orthostatic intolerance test, with the absence of orthostatic hypotension (a decrease in systolic blood pressure (BP) of ~20 or more mmHg with or without a decrease in diastolic BP of ~10 or more mmHg). There are common conditions/comorbidities that may occur which may not be physiologically explained by the orthostatic intolerance or tachycardia. These symptoms include chronic fatigue, dizziness, syncope, migraines, GI disorders, nausea, fibromyalgia, joint hypermobility, and cognitive disturbances. As a result of this uncertainty with treatment, and wanting what is best for the patient in the long run, this poses the following question: In adult women (18-45 years old) diagnosed with POTS who experience comorbidities of daily fatigue and “brain fog” [P], will stimulants be the most beneficial treatment for these symptoms [I], as opposed to the current standard of care (exercise) [C] for symptomatic relief [O]? This study will evaluate different clinical trials and studies that look at the effects of stimulants in POTS management versus exercise and the outcomes of these interventions. Methods: A literature search was conducted on Pubmed, Google Scholar, and Ovid, with all of the articles found being from Pubmed. A randomized control trial, two longitudinal studies, a questionnaire, and two retroactive studies were chosen to be analyzed and compared. These studies were chosen based on which were most relevant to this analysis. Results: The evidence collected yielded some confounding information. One study compared modafinil to a placebo, and this did not yield any statistically significant results of modafinil having better outcomes than a placebo. Two studies were longitudinal studies which followed POTS patients completing exercise programs. These showed statistically significant improvement in the symptoms of the patients that were able to complete these programs. Two retrospective studies aimed at studying if stimulants were helpful to patients with cognitive decline. These studies found that it did help these patients subjectively, however the collection and analysis of results in these studies were unreliable. Discussion: Each of the studies had differing outcome measures, so comparing them was a difficult process. Only one study used blinding and it would have been ideal to have more of these studies available. There was lack of long term data/follow up, which is another area which needs to be researched more extensively. The use of an exercise program was statistically significant in improving the symptoms and quality of life of POTS patients. There were not any negative health outcomes reported for any of the interventions (exercise or stimulant therapy), but long term follow-ups were not completed. Further studies of these topics is necessary for clinical application of stimulant medications. Conclusion: Stimulant therapy has been shown to help cognitive function in those with ADHD, but no studies have confirmed its benefit in those with POTS who experience cognitive impairment. It is important to use the least amount of medication for the maximum benefit in these patients because they often get prescribed multiple medications to treat symptoms because they can be very bothersome. Medications come with side effects, and when patients are on multiple medications, it may be difficult to know which symptoms are from the disease or from medication. More research needs to be done in the utilization of stimulant medication in the POTS population. Exercise has shown to be effective at improving the quality of life of those with this disease. Further research is necessary to find the most beneficial treatment for these patients that they will be compliant with

Induction of terpene biosynthesis in dinoflagellate symbionts of Caribbean gorgonians

abstract: This report describes a series of experiments designed to determine if terpene biosynthesis is inducible in two families of marine terpenes, pseudopterosins from the gorgonian coral Pseudopterogorgia elisabethae and fuscol from Eunicea fusca. Since we have recently shown that terpene biosynthesis is not under the control of the invertebrate host, but rather occurs within a dinoflagellate preparation, we examined the terpene content of the dinoflagellate symbiont following a decrease in UV/vis radiation as well as in response to the addition of methyl jasmonate, salicylic acid and gibberellic acid. We demonstrated that pseudopterosin and fuscol biosynthesis can be markedly increased through the addition of the plant bioactive substances. We also demonstrated that, while the terpene content of P. elisabethae increases in response to decreased UV/vis light, this is due primarily to an increase in the concentration of the dinoflagellate rather than simply an induction of terpene biosynthesis

Comparison of two total RNA extraction protocols using the marine gorgonian coral Pseudopterogorgia elisabethae and its symbiont Symbiodinium sp.

Marine invertebrates such as soft corals are important sources of secondary metabolites with promising biomedical applications and commercial value. RNA isolation in conjunction with reverse-transcriptase polymerase chain reaction (RT-PCR) are valuable tools utilized to study the molecular elements involved in secondary metabolite production and functional genomics. Two total RNA extraction protocols were compared using fresh tissue and flash frozen preparations from the coral Pseudopterogorgia elisabethae and from its symbiont Symbiodinium sp. isolated using RNeasy minicolumns (Qiagen®) and Trizol reagent (Invitrogen®). In general, higher yields were obtained by using Trizol reagent when compared to RNeasy. No significant differences were observed in RNA yield when live or flash frozen tissue was used. However, flash frozen holobiont tissue isolated by Trizol resulted in the highest RNA yield of all preparations analyzed. To conclude, both protocols are suitable for RNA isolation. Trizol is recommended if higher yields are the primary concern, but RNeasy is recommended if time is an issue

Leave your Stamp: Reconstruction of the Scarab Production Chain

Scarabs are beetle-shaped seals originating in Egypt during the third millennium BC. They were first imported into the southern Levant during the early second millennium BC (Middle Bronze Age I), leading to a flourishing of local production in the following centuries. From this point until the late first millennium BC, scarabs were the most common form of stamp-seal in the southern Levant. Past scholarship attempted to create criteria for the identification of Egyptian vs. southern Levantine scarabs, emphasizing mostly their typological and iconographic features, while other aspects of their production have largely been disregarded. In this article, we present the results of an experiment conducted to study a plausible production sequence involved in scarab manufacture. The broad goal of the experiment is to understand the various methods of production and to place the phases of production in a sequential order (their chaîne opératoire)